Analysis of mitochondrial carrier structure and function: Mitochondrial carriers are proteins of the mitochondrial inner membrane that exchange metabolites between the cytosol and the matrix. They are key players in respiration, the TCA cycle, the urea cycle and fatty acid metabolism. Current results from genome sequencing projects suggest there are nearly 50 carriers in yeast and close to 200 in C. elegans. As such, they form a moderately sized protein family of critical importance for eucaryotes. The ADP/ATP carrier is one of the most thoroughly studied members of this group. It serves as an archetype for the carrier superfamily. Yeast AAC2, is an ideal model system for analysis of carrier structure and function. Mutants expressed in a knockout strain can be purified and reconstituted to measure transport parameters, and powerful genetic methods such as selection for revertant mutations can be performed. This system has already been employed to identify eight essential residues in the molecule and 22 second site revertant mutations. Four of these revertants are indicative of charge pairs in the ADP/ATP carrier that would not have been proposed on any biochemical basis. This proposal will extend these results by continuing the search for charge pairs predicted to exist in the ADP/ATP carrier and probably most other mitochondrial carriers as well. In keeping with the archetype role of the ADP/ATP carrier, mutants already made will be used to resolve a major unanswered question about these carriers. Biophysical studies have shown the ADP/ATP carrier and other carriers are dimers. Each subunit contains six transmembrane segments, but no one knows whether the translocation pathway forms at the dimer interface, or independently within each subunit The genetics of yeast can answer this question by expressing two non-functional AAC2 mutants on separate plasmids in the knockout host. We already know that when D149S and R252T are expressed on a single plasmid they complement one another and produce a functional protein. If they complement one another in separate proteins, then the translocation pathway must form at the dimer interface, and not within two single subunits. This question will be answered. As some of the larger scale questions become solved, the ADP/ATP carrier in yeast will permit higher resolution analysis. By engineering a cysteine free AAC2 protein, it will be possible to map the helical contacts between the six transmembrane segments of the monomer and the equally interesting contacts between helices of the dimer. Once a cysteine free protein is available, cysteines can be placed anywhere in the sequence to map helix interactions by disulfide bond formation. Successful engineering of specific disulfide bonds should identify which helices are adjacent in the structure and which helical surfaces are in contact. Finally, the matter of human disease caused by mutations in the ADP/ATP carrier is addressed. It seems clear that mild mutations of this protein would lead to mitochondrial insufficiency that should manifest itself in disease, specifically diseases that are the consequence of poor energy utilization such as myopathies. A PCR based screen is proposed to search for these mutations in patients with possible mitochondrial defects.